Pyrimidine substituted purine compounds as kinase (S) inhibitors

ABSTRACT

The present invention relates to a purine compound useful as a kinase inhibitor. The compound has the structure: 
     
       
         
         
             
             
         
       
         
         
           
             or a pharmaceutically acceptable salt thereof.

The instant application, filed on 7 May 2014, is a continuation of U.S.patent application Ser. No. 13/262,544, filed on 27 Dec. 2011, which isa National Stage entry of International Patent ApplicationPCT/SG2009/000124, filed 3 Apr. 2009.

FIELD

The present invention relates to5-(9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylamine,methods for its preparation, pharmaceutical compositions containing thiscompound and uses of this compound in the treatment of certain kinaserelated disorders/conditions.

BACKGROUND

The search for kinase inhibitors has proven to be a fruitful area forthe development of useful pharmaceutically active substances. Kinases,which are alternatively known as phosphotransferases, are enzymes thattransfer phosphate groups from high energy donor molecules (for exampleATP) to specific target molecules (typically called substrates) in aprocess termed phosphorylation. One of the largest groups of kinases arethe protein kinases which act on and modify the activity of specificproteins.

As a result of the potential of kinase inhibitors to act aspharmaceutically active compounds a significant amount of research hasbeen carried out to discover compounds that display appropriate activityagainst these targets. In the cancer area two kinases that haveattracted attention as potential targets for therapeutic compoundsinclude mTOR and PI3. An example of research in this area is thatdisclosed in PCT/SG2008/000379 which discloses a number of compoundshaving kinase activity against both mTOR and PI3.

Compounds that inhibit both mTOR and PI3 kinases simultaneously may beexpected to provide powerful anti-proliferative, anti-angiogenic andantitumor activity since these compounds act at multiple points in thePI3K/Akt/mTOR pathway. A number of inhibitors of this type are now beinginvestigated in a clinical setting for the first time (e.g. BEZ235,XL765, GDC0941, PX866, SF1126).

In the search for suitable drug candidates a number of factors are takeninto consideration in the final determination of whether a compound is asuitable drug candidate or not. Accordingly in making an assessment of apotential compound for further development a number of factors are takeninto consideration in addition to the primary inhibitory activity of thecompound per se. In making this assessment the skilled medicinal chemistlooks at the “drug like properties” of the molecule and includes anassessment of factors such as its activity against the target ofinterest, the solubility of the compounds of interest (if they are notsoluble they typically make poor drug candidates), the metabolicstability of the compound in vitro and in vivo, and the potential sideeffects that could be caused by the compound on the body amongst others.The present applicants have identified a compound with significantlyimproved drug like properties in comparison with other compounds in thearea.

SUMMARY

The present invention provides a compound of the formula (I):

or a pharmaceutically acceptable salt thereof.

In addition to the compound of Formula I, the embodiments disclosed arealso directed to pharmaceutically acceptable salts, pharmaceuticallyacceptable N-oxides, pharmaceutically acceptable prodrugs, andpharmaceutically active metabolites of the compound, andpharmaceutically acceptable salts of such metabolites.

The invention also relates to pharmaceutical compositions including thecompound of the invention with a pharmaceutically acceptable carrier,diluent or excipient.

In a further aspect the invention provides a method of inhibiting aprotein kinase selected from the group consisting of a serine/threonineprotein kinase or a fragment or a complex thereof or a functionalequivalent thereof and a PI3 kinase or a fragment or a complex thereofor a functional equivalent thereof, the method including exposing theprotein kinase or a fragment or complex thereof or a functionalequivalent thereof and/or co-factor(s) thereof to an effective amount ofa compound of the invention.

The compound disclosed herein may act directly and solely on the kinasemolecule or a complex or fragment thereof to inhibit biologicalactivity. However, it is understood that the compound may also act atleast partially on co-factors that are involved in the phosphorylationprocess. Known kinase co-factors include ionic species (such as zinc andcalcium), lipids (such as phosphatidylserine), and diacylglycerols.

In some embodiments the protein kinase is a serine/threonine proteinkinase or a fragment or a complex thereof or a functional equivalentthereof. In some embodiments the serine/threonine protein kinase or afragment or complex thereof is an mTOR protein kinase or a fragmentthereof, or a complex thereof or a functional equivalent thereof. Insome embodiments the serine/threonine protein kinase is mTORC1 or afragment or complex thereof or a functional equivalent thereof. In someembodiments the serine/threonine protein kinase is mTORC2 or a fragmentor complex thereof or a functional equivalent thereof.

In some embodiments the protein kinase is a PI3 kinase or a fragmentthereof or a complex thereof or a functional equivalent thereof. In someembodiments the PI3 kinase or a fragment thereof or a complex thereof ora functional equivalent thereof, is a class I PI3K or a fragment thereofor a complex thereof or a functional equivalent thereof.

In one embodiment of the method exposing the one or more proteinkinase(s) to the compound includes administering the compound to amammal containing the one or more protein kinase(s).

In an even further aspect the invention provides the use of a compoundof the invention to inhibit one or more protein kinase(s) selected fromthe group consisting of a serine/threonine protein kinase or a fragmentor a complex thereof or a functional equivalent thereof and a PI3 kinaseor a fragment or a complex thereof or a functional equivalent thereof.

In some embodiments the protein kinase is a serine/threonine proteinkinase or a fragment or a complex thereof or a functional equivalentthereof. In some embodiments the serine/threonine protein kinase or afragment or complex thereof is an mTOR protein kinase or a fragmentthereof, or a complex thereof or a functional equivalent thereof. Insome embodiments the serine/threonine protein kinase is mTORC1 or afragment or complex thereof or a functional equivalent thereof. In someembodiments the serine/threonine protein kinase is mTORC2 or a fragmentor complex thereof or a functional equivalent thereof.

In some embodiments the protein kinase is a PI3 kinase or a fragmentthereof or a complex thereof or a functional equivalent thereof. In someembodiments the PI3 kinase or a fragment thereof or a complex thereof ora functional equivalent thereof, is a class I PI3K or a fragment thereofor a complex thereof or a functional equivalent thereof.

In an even further aspect the invention provides a method of treating orpreventing a condition in a mammal in which inhibition of one or moreprotein kinase(s) selected from the group consisting of aserine/threonine protein kinase or a fragment or a complex thereof or afunctional equivalent thereof and a PI3 kinase or a fragment or acomplex thereof or a functional equivalent thereof, prevents, inhibitsor ameliorates a pathology or a symptomology of the condition, themethod including administration of a therapeutically effective amount ofa compound of the invention.

In some embodiments the protein kinase is a serine/threonine proteinkinase or a fragment or a complex thereof or a functional equivalentthereof. In some embodiments the serine/threonine protein kinase or afragment or complex thereof is an mTOR protein kinase or a fragmentthereof, or a complex thereof or a functional equivalent thereof. Insome embodiments the serine/threonine protein kinase is mTORC1 or afragment or complex thereof or a functional equivalent thereof. In someembodiments the serine/threonine protein kinase is mTORC2 or a fragmentor complex thereof or a functional equivalent thereof.

In some embodiments the protein kinase is a PI3 kinase or a fragmentthereof or a complex thereof or a functional equivalent thereof. In someembodiments the PI3 kinase or a fragment thereof or a complex thereof ora functional equivalent thereof, is a class I PI3K or a fragment thereofor a complex thereof or a functional equivalent thereof.

In some embodiments the condition is cancer. In some embodiments thecancer is selected from the group consisting of Hematologic cancer suchas myeloproliferative disorders (idiopathic myelofibrosis, polycythemiavera, essential thrombocythemia, chronic myeloid leukemia), myeloidmetaplasia, chronic myelomonocytic leukemia, acute lymphocytic leukemia,acute erythroblastic leukemia, Hodgkin's and Non Hodgkin's disease,B-cell lymphoma, acute T-cell leukemia, myelodysplastic syndromes,plasma cell disorder, hairy cell leukemia, kaposi's sarcoma, lymphomaand hyperproliferative conditions such as psoriasis and restenosis;gynaecologic cancer such as breast carcinoma, ovarian cancer, cervicalcancer, vaginal and vulva cancer, endometrial hyperplasia;gastrointestinal tract cancer such as colorectal carcinoma, polyps,liver cancer, gastric cancer, pancreatic cancer, gall bladder cancer;urinary tract cancer such as prostate cancer, kidney and renal cancer;urinary bladder cancer, urethral cancer, penile cancer; skin cancer suchas melanoma; brain tumour such as glioblastoma, neuroblastoma,astrocytoma, ependynoma, brain-stem gliomas, medulloblastoma,menigiomas, astrocytoma, oligodendroglioma; head and neck cancer such asnasopharyngeal carcinoma, laryngeal carcinoma; respiratory tract cancersuch as lung carcinoma (NSCLC and SCLC), mesothelioma; eye disease suchas retinoblastoma; musculo-skeleton diseases such as osteosarcoma,musculoskeleletal neoplasm; Squamous cell carcinoma and fibroid tumour.In other embodiments, the compound of this invention can be used totreat pre-cancer conditions or hyperplasia including familialadenomatous polyposis, colonic adenomatous polyps, myeloid dysplasia,endometrial dysplasia, endometrial hyperplasia with atypia, cervicaldysplasia, vaginal intraepithelial neoplasia, benign prostatichyperplasia, papillomas of the larynx, actinic and solar keratosis,seborrheic keratosis and keratoacanthoma.

In some embodiments the condition is an autoimmune or inflammatorydisease or a disease supported by excessive neovascularisation. Diseasesthat have been attributed with some degree of autoimmune etiology, orthat involve pathological inflammatory and neovascularization responses,include the following: acute disseminated encephalomyelitis, Addison'sdisease, agammaglobulinemia, agranulocytosis, allergic asthma, allergicencephalomyelitis, allergic rhinitis, alopecia areata, alopecia senilis,anerythroplasia, ankylosing spondylitis, antiphospholipid antibodysyndrome, aortitis syndrome, aplastic anemia, atopic dermatitis,autoimmune haemolytic anemia, autoimmune hepatitis, autoimmuneoophoritis, Balo disease, Basedow's disease, Behcet's disease, bronchialasthma, Castleman's syndrome, celiac disease, Chagas disease, chronicinflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,Cogans syndrome, cornical cornea, comical leukoma, Coxsackiemyocarditis, CREST disease, Crohn's disease, cutaneous eosinophilia,cutaneous T-cell lymphoma, dermatitis erythrema multiforme,dermatomyositis, diabetic retinopathy, Dressler's syndrome, dystrophiaepithelialis corneae, eczematous dermatitis, endometriosis, eosinophilicfasciitis, eosinophilic gastroenteritis, epidermolysis bullosa, Evanssyndrome, fibrosing alveolitis, gestational pemphigoid,glomerulonephritis, Goodpasture's syndrome, graft-versus-host disease,Graves' disease, Guillain-Barre Syndrome, Hashimoto's disease,haemolytic-uretic syndrome, herpetic keratitis, ichthyosis vulgaris,idiopathic intersititial pneumonia, idiopathic thrombocytopenic purpura,inflammatory bowel diseases, Kawasaki's disease, keratitis,keratoconjunctivitis, Lambert-Eaton syndrome, leukoderma vulgaris,lichen planus, lichen sclerosus, Lyme disease, linear IgA disease,macular degeneration, megaloblastic anemia, Meniere's disease, Mooren'sulcer, Mucha-Habermann disease, multiple myositis, multiple sclerosis,myasthenia gravis, necrotizing enterocolitis, neuromyelitis optica,ocular pemphigus, opsoclonus myoclonus syndrome, Ord's thyroiditis,paroxysmal nocturnal hemoglobinuria, Parsonnage-Turner syndrome,pemphigus, periodontitis, pernicious anemia, pollen allergies,polyglandular autoimmune syndrome, posterior uveitis, primary biliarycirrhosis, proctitis, pseudomembranous colitis, psoriasis, pulmonaryemphysema, pyoderma, Reiter's syndrome, reversible obstructive airwaydisease, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis,Sezary's syndrome, Sjogren's syndrome, subacute bacterial endocarditis,systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis,Tolosa-Hunt syndrome, Type I diabetes mellitus, ulcerative colitis,urticaria, vernal conjunctivitis, vitiligo, Vogy-Koyanagi-Haradasyndrome and Wegener's granulomatosis. In some embodiments the conditionis endometriosis.

In an even further aspect the invention provides use of a compound ofthe invention in the preparation of a medicament for treating acondition in an animal in which inhibition of one or more proteinkinase(s) selected from the group consisting of a serine/threonineprotein kinase or a fragment or a complex thereof or a functionalequivalent thereof and a PI3 kinase or a fragment or a complex thereofor a functional equivalent thereof, prevents, inhibits or ameliorates apathology or a symptomology of the condition.

In another aspect the present invention provides the use of a compoundof the invention or a pharmaceutically acceptable salt, N-oxide orprodrug thereof in the treatment of a condition in which inhibition ofone or more protein kinase(s) selected from the group consisting of aserine/threonine protein kinase or a fragment or a complex thereof or afunctional equivalent thereof and a PI3 kinase or a fragment or acomplex thereof or a functional equivalent thereof, prevents, inhibitsor ameliorates a pathology or a symptomology of the condition

In some embodiments the protein kinase is a serine/threonine proteinkinase or a fragment or a complex thereof or a functional equivalentthereof. In some embodiments the serine/threonine protein kinase or afragment or complex thereof is an mTOR protein kinase or a fragmentthereof, or a complex thereof or a functional equivalent thereof. Insome embodiments the serine/threonine protein kinase is mTORC1 or afragment or complex thereof or a functional equivalent thereof. In someembodiments the serine/threonine protein kinase is mTORC2 or a fragmentor complex thereof or a functional equivalent thereof.

In some embodiments the protein kinase is a PI3 kinase or a fragmentthereof or a complex thereof or a functional equivalent thereof. In someembodiments the PI3 kinase or a fragment thereof or a complex thereof ora functional equivalent thereof, is a class I PI3K or a fragment thereofor a complex thereof or a functional equivalent thereof.

In another aspect the present invention provides a method of preventionor treatment of a proliferative condition in a subject, the methodincluding administration of a therapeutically effective amount of acompound of the invention.

In another aspect the present invention provides the use of a compoundof the invention in the preparation of a medicament for treating aproliferative condition in a subject.

In some embodiments the condition is cancer. In some embodiments thecancer is selected from the group consisting of Hematologic cancer suchas myeloproliferative disorders (idiopathic myelofibrosis, polycythemiavera, essential thrombocythemia, chronic myeloid leukemia), myeloidmetaplasia, chronic myelomonocytic leukemia, acute lymphocytic leukemia,acute erythroblastic leukemia, Hodgkin's and Non Hodgkin's disease,B-cell lymphoma, acute T-cell leukemia, myelodysplastic syndromes,plasma cell disorder, hairy cell leukemia, kaposi's sarcoma, lymphoma;gynaecologic cancer such as breast carcinoma, ovarian cancer, cervicalcancer, vaginal and vulva cancer, endometrial hyperplasia;gastrointestinal tract cancer such as colorectal carcinoma, polyps,liver cancer, gastric cancer, pancreatic cancer, gall bladder cancer;urinary tract cancer such as prostate cancer, kidney and renal cancer;urinary bladder cancer, urethral cancer, penile cancer; skin cancer suchas melanoma; brain tumour such as glioblastoma, neuroblastoma,astrocytoma, ependynoma, brain-stem gliomas, medulloblastoma,menigiomas, astrocytoma, oligodendroglioma; head and neck cancer such asnasopharyngeal carcinoma, laryngeal carcinoma; respiratory tract cancersuch as lung carcinoma (NSCLC and SCLC), mesothelioma; eye disease suchas retinoblastoma; musculo-skeleton diseases such as osteosarcoma,musculoskeleletal neoplasm; Squamous cell carcinoma and fibroid tumour.In some embodiments the condition is endometriosis.

Additionally, Formula (I) is intended to cover, where applicable,solvated as well as unsolvated forms of the compound. Thus, each formulaincludes compounds having the indicated structure, including thehydrated as well as the non-hydrated forms.

These and other features of the present teachings are set forth herein.

DETAILED DESCRIPTION

In this specification a number of terms are used which are well known toa skilled addressee. Nevertheless for the purposes of clarity a numberof terms will be defined.

The term “pharmaceutically acceptable salts” refers to salts that retainthe desired biological activity of the above-identified compound, andinclude pharmaceutically acceptable acid addition salts and baseaddition salts. Suitable pharmaceutically acceptable acid addition saltsof compound of Formula (I) may be prepared from an inorganic acid orfrom an organic acid. Examples of such inorganic acids are hydrochloric,sulfuric, and phosphoric acid. Appropriate organic acids may be selectedfrom aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic andsulfonic classes of organic acids, examples of which are formic, acetic,propionic, succinic, glycolic, gluconic, lactic, malic, tartaric,citric, fumaric, maleic, alkyl sulfonic, arylsulfonic. Additionalinformation on pharmaceutically acceptable salts can be found inRemington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co.,Easton, Pa. 1995. In the case of agents that are solids, it isunderstood by those skilled in the art that the inventive compound,agents and salts may exist in different crystalline or polymorphicforms, all of which are intended to be within the scope of the presentinvention and specified formulae.

The term “therapeutically effective amount” or “effective amount” is anamount sufficient to effect beneficial or desired clinical results. Aneffective amount can be administered in one or more administrations. Aneffective amount is typically sufficient to palliate, ameliorate,stabilize, reverse, slow or delay the progression of the disease state.

The term “functional equivalent” is intended to include variants of thespecific protein kinase species described herein. It will be understoodthat kinases may have isoforms, such that while the primary, secondary,tertiary or quaternary structure of a given kinase isoform is differentto the prototypical kinase, the molecule maintains biological activityas a protein kinase. Isoforms may arise from normal allelic variationwithin a population and include mutations such as amino acidsubstitution, deletion, addition, truncation, or duplication. Alsoincluded within the term “functional equivalent” are variants generatedat the level of transcription. Other functional equivalents includekinases having altered post-translational modification such asglycosylation.

The compound of the invention displays superior drug like propertieswhich are described in greater detail below, in comparison tostructurally similar compounds in the area. These superior propertiessuggest that the compound of the invention may well be the compound ofchoice as a pharmaceutical development candidate in the area. As a firstobservation the compound of the invention displays comparable if notsuperior activity in the inhibition of the two kinases of interestnamely mTOR and PI3. The activity of the compound of the inventionagainst PI3 is stronger than all comparator compounds tested and itdisplays activity against mTOR that is both comparable to comparatorcompounds as well as being at an acceptable level for therapeuticapplications.

Notwithstanding that the enzyme activity tests indicate that almost allcomparator compounds had acceptable activity levels the further testingof the compounds indicated that a number could be ruled out aspharmaceutical development candidates on other grounds. Thus for examplethe compound of the invention had a reasonable aqueous solubility level(178 μM) indicating that it can be formulated into an orally absorbablepharmaceutical formulation whereas a number of the comparator compoundsdid not demonstrate acceptable solubility. As such the compound of theinvention displayed the combination of excellent activity and acceptablesolubility characteristics.

Of the compounds that displayed the combination of activity andsolubility the compound of the invention was far superior in itsmetabolic stability properties. The compound of the invention hadexcellent stability in human liver microsome studies indicating that itwas robust and relatively resistant to degradation in the physiologicalenvironment. In contrast the other compounds that displayed thecombination of activity and solubility were nowhere near as stable inthese studies. As such the compound of the present inventiondemonstrates a unique combination of activity, solubility and stabilitythat make it superior as a drug candidate in comparison to relatedcompounds in the area notwithstanding the apparent close structuralsimilarity of some of these compounds.

The compound of the invention has the ability to inhibit the activity ofcertain protein kinases. The ability to inhibit kinase activity may be aresult of the compound of the invention acting directly and solely onthe kinase molecule to inhibit biological activity. However, it isunderstood that the compound may also act at least partially onco-factors of the kinase in question that are involved in thephosphorylation process. The compound may have activity against PI3protein kinases or a fragment or a complex or a functional equivalentthereof. The compound may have activity against certain serine/threoninekinases such as mTOR or a fragment or complex or functional equivalentthereof.

The inhibition of the protein kinase may be carried out in any of anumber of well known ways in the art. For example if inhibition of theprotein kinase in vitro is desired an appropriate amount of the compoundof the invention may be added to a solution containing the purifiedkinase enzyme. In circumstances where it is desired to inhibit theactivity of the kinase in a mammal the inhibition of the kinasetypically involves administering the compound to a mammal containing thekinase.

Accordingly the compound of the invention may find a multiple number ofapplications in which their ability to inhibit protein kinases of thetype mentioned above can be utilised. For example the compounds may beused to inhibit serine/threonine protein kinases. The compounds may alsobe used in treating or preventing a condition in a mammal in whichinhibition of a protein kinase and/or co-factor thereof prevents,inhibits or ameliorates a pathology or a symptomology of the condition.

The compound disclosed has the ability to be used in the treatment ofproliferative disorders. An example of such a disorder is cancer. It isanticipated that the compounds will have the ability to treat both solidand liquid tumors. In some embodiments the cancers that may be treatedby a compound of the present invention include solid tumors andhematological cancers.

As used herein, the term “cancer” is a general term intended toencompass the vast number of conditions that are characterized byuncontrolled abnormal growth of cells. It is anticipated that thecompounds of the invention will be useful in treating various cancersincluding but not limited to bone cancers, brain and CNS tumours, breastcancers, colorectal cancers, endocrine cancers including adrenocorticalcarcinoma, pancreatic cancer, pituitary cancer, thyroid cancer,parathyroid cancer, thymus cancer, gastrointestinal cancers, Livercancer, extra hepatic bile duct cancer, gastrointestinal carcinoidtumour, gall bladder cancer, genitourinary cancers, gynaecologicalcancers, head and neck cancers, leukemias, myelomas, hematologicaldisorders, lung cancers, lymphomas, eye cancers, skin cancers, softtissue sarcomas, adult soft tissue sarcoma, Kaposi's sarcoma, urinarysystem cancers.

Exemplary cancers that may be treated by compounds of this inventioninclude Hematologic cancer such as myeloproliferative disorders(idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia,chronic myeloid leukemia), myeloid metaplasia, chronic myelomonocyticleukemia, acute lymphocytic leukemia, acute erythroblastic leukemia,Hodgkin's and Non Hodgkin's disease, B-cell lymphoma, acute T-cellleukemia, myelodysplastic syndromes, plasma cell disorder, hairy cellleukemia, kaposi's sarcoma, lymphoma and hyperproliferative conditionssuch as psoriasis and restenosis; gynaecologic cancer such as breastcarcinoma, ovarian cancer, cervical cancer, vaginal and vulva cancer,endometrial hyperplasia; gastrointestinal tract cancer such ascolorectal carcinoma, polyps, liver cancer, gastric cancer, pancreaticcancer, gall bladder cancer; urinary tract cancer such as prostatecancer, kidney and renal cancer; urinary bladder cancer, urethralcancer, penile cancer; skin cancer such as melanoma; brain tumour suchas glioblastoma, neuroblastoma, astrocytoma, ependynoma, brain-stemgliomas, medulloblastoma, menigiomas, astrocytoma, oligodendroglioma;head and neck cancer such as nasopharyngeal carcinoma, laryngealcarcinoma; respiratory tract cancer such as lung carcinoma (NSCLC andSCLC), mesothelioma; eye disease such as retinoblastoma;musculo-skeleton diseases such as osteosarcoma, musculoskeleletalneoplasm; Squamous cell carcinoma and fibroid tumour. Compounds of thisinvention may also be used to treat pre-cancer conditions or hyperplasiaincluding familial adenomatous polyposis, colonic adenomatous polyps,myeloid dysplasia, endometrial dysplasia, endometrial hyperplasia withatypia, cervical dysplasia, vaginal intraepithelial neoplasia, benignprostatic hyperplasia, papillomas of the larynx, actinic and solarkeratosis, seborrheic keratosis and keratoacanthoma.

It is also anticipated that the compound of the invention will be usefulin treating autoimmune or inflammatory diseases or diseases supported byexcessive neovascularisation. Diseases that have been attributed withsome degree of autoimmune etiology, or that involve pathologicalinflammatory and neovascularization responses, include, but are notlimited to, the following: acute disseminated encephalomyelitis,Addison's disease, agammaglobulinemia, agranulocytosis, allergic asthma,allergic encephalomyelitis, allergic rhinitis, alopecia areata, alopeciasenilis, anerythroplasia, ankylosing spondylitis, antiphospholipidantibody syndrome, aortitis syndrome, aplastic anemia, atopicdermatitis, autoimmune haemolytic anemia, autoimmune hepatitis,autoimmune oophoritis, Balo disease, Basedow's disease, Behcet'sdisease, bronchial asthma, Castleman's syndrome, celiac disease, Chagasdisease, chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, Cogans syndrome, cornical cornea, cornicalleukoma, Coxsackie myocarditis, CREST disease, Crohn's disease,cutaneous eosinophilia, cutaneous T-cell lymphoma, dermatitis erythremamultiforme, dermatomyositis, diabetic retinopathy, Dressler's syndrome,dystrophia epithelialis corneae, eczematous dermatitis, eosinophilicfasciitis, eosinophilic gastroenteritis, epidermolysis bullosa, Evanssyndrome, fibrosing alveolitis, gestational pemphigoid,glomerulonephritis, Goodpasture's syndrome, graft-versus-host disease,Graves' disease, Guillain-Barre Syndrome, Hashimoto's disease,haemolytic-uretic syndrome, herpetic keratitis, ichthyosis vulgaris,idiopathic intersititial pneumonia, idiopathic thrombocytopenic purpura,inflammatory bowel diseases, Kawasaki's disease, keratitis,keratoconjunctivitis, Lambert-Eaton syndrome, leukoderma vulgaris,lichen planus, lichen sclerosus, Lyme disease, linear IgA disease,macular degeneration, megaloblastic anemia, Meniere's disease, Mooren'sulcer, Mucha-Habermann disease, multiple myositis, multiple sclerosis,myasthenia gravis, necrotizing enterocolitis, neuromyelitis optica,ocular pemphigus, opsoclonus myoclonus syndrome, Ord's thyroiditis,paroxysmal nocturnal hemoglobinuria, Parsonnage-Turner syndrome,pemphigus, periodontitis, pernicious anemia, pollen allergies,polyglandular autoimmune syndrome, posterior uveitis, primary biliarycirrhosis, proctitis, pseudomembranous colitis, psoriasis, pulmonaryemphysema, pyoderma, Reiter's syndrome, reversible obstructive airwaydisease, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis,Sezary's syndrome, Sjogren's syndrome, subacute bacterial endocarditis,systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis,Tolosa-Hunt syndrome, Type I diabetes mellitus, ulcerative colitis,urticaria, vernal conjunctivitis, vitiligo, Vogy-Koyanagi-Haradasyndrome and Wegener's granulomatosis. In some embodiments the conditionis endometriosis.

The compound of the invention may also be used the preparation of amedicament for treating a condition in an animal in which inhibition ofa protein kinase can prevent, inhibit or ameliorate the pathology orsymptomology of the condition. The compound of the invention may also beused in the preparation of a medicament for the treatment or preventionof a kinase-related disorder.

Administration of the compound of Formula (I) to humans can be by any ofthe accepted modes for enteral administration such as oral or rectal, orby parenteral administration such as subcutaneous, intramuscular,intravenous and intradermal routes. Injection can be bolus or viaconstant or intermittent infusion. The active compound is typicallyincluded in a pharmaceutically acceptable carrier or diluent and in anamount sufficient to deliver to the patient a therapeutically effectivedose. In various embodiments the inhibitor compound may be selectivelytoxic or more toxic to rapidly proliferating cells, e.g. canceroustumours, than to normal cells.

In using the compound of the invention it can be administered in anyform or mode which makes the compound bioavailable. One skilled in theart of preparing formulations can readily select the proper form andmode of administration depending upon the particular characteristics ofthe compound selected, the condition to be treated, the stage of thecondition to be treated and other relevant circumstances. We refer thereader to Remingtons Pharmaceutical Sciences, 19^(th) edition, MackPublishing Co. (1995) for further information.

The compound of the present invention can be administered alone or inthe form of a pharmaceutical composition in combination with apharmaceutically acceptable carrier, diluent or excipient. The compoundof the invention, while effective itself, is typically formulated andadministered in the form of their pharmaceutically acceptable salts asthese forms are typically more stable, more easily crystallised and haveincreased solubility.

The compound is, however, typically used in the form of pharmaceuticalcompositions which are formulated depending on the desired mode ofadministration. As such in some embodiments the present inventionprovides a pharmaceutical composition including a compound of theinvention and a pharmaceutically acceptable carrier, diluent orexcipient. The compositions are prepared in manners well known in theart.

The invention in other embodiments provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention. In sucha pack or kit can be found a container having a unit dosage of theagent(s). The kits can include a composition comprising an effectiveagent either as concentrates (including lyophilized compositions), whichcan be diluted further prior to use or they can be provided at theconcentration of use, where the vials may include one or more dosages.Conveniently, in the kits, single dosages can be provided in sterilevials so that the physician can employ the vials directly, where thevials will have the desired amount and concentration of agent(s).Associated with such container(s) can be various written materials suchas instructions for use, or a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The compound of the invention may be used or administered in combinationwith one or more additional drug(s) for the treatment of thedisorder/diseases mentioned. The components can be administered in thesame formulation or in separate formulations. If administered inseparate formulations the compounds of the invention may be administeredsequentially or simultaneously with the other drug(s).

In addition to being able to be administered in combination with one ormore additional drugs, the compound of the invention may be used in acombination therapy. When this is done the compound is typicallyadministered in combination with each other. Thus the compound of theinvention may be administered either simultaneously (as a combinedpreparation) or sequentially in order to achieve a desired effect. Thisis especially desirable where the therapeutic profile of each compoundis different such that the combined effect of the two drugs provides animproved therapeutic result.

Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils(such as olive oil), and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservative,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of micro-organisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents that delay absorptionsuch as aluminium monostearate and gelatin.

If desired, and for more effective distribution, the compounds can beincorporated into slow release or targeted delivery systems such aspolymer matrices, liposomes, and microspheres.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

The active compound can also be in microencapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminiummetahydroxide, bentonite, agar-agar, and tragacanth, and mixturesthereof.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat room temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active compound.

Dosage forms for topical administration of a compound of this inventioninclude powders, patches, sprays, ointments and inhalants. The activecompound is mixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives, buffers, or propellantswhich may be required.

The amount of compound administered will preferably treat and reduce oralleviate the condition. A therapeutically effective amount can bereadily determined by an attending diagnostician by the use ofconventional techniques and by observing results obtained underanalogous circumstances. In determining the therapeutically effectiveamount a number of factors are to be considered including but notlimited to, the species of animal, its size, age and general health, thespecific condition involved, the severity of the condition, the responseof the patient to treatment, the particular compound administered, themode of administration, the bioavailability of the preparationadministered, the dose regime selected, the use of other medications andother relevant circumstances.

A preferred dosage will be a range from about 0.01 to 300 mg perkilogram of body weight per day. A more preferred dosage will be in therange from 0.1 to 100 mg per kilogram of body weight per day, morepreferably from 0.2 to 80 mg per kilogram of body weight per day, evenmore preferably 0.2 to 50 mg per kilogram of body weight per day. Asuitable dose can be administered in multiple sub-doses per day.

Synthesis of the Compound of the Invention

5-(9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylaminewas prepared from dichloropurine using a 5 step procedure depicted inscheme 1.

EXAMPLES

In the examples described below, unless otherwise indicated, alltemperatures in the following description are in degrees Celsius and allparts and percentages are by weight, unless indicated otherwise.

Various starting materials and other reagents were purchased fromcommercial suppliers, such as Aldrich Chemical Company or LancasterSynthesis Ltd., and used without further purification, unless otherwiseindicated. Tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) werepurchased from Aldrich in SureSeal bottles and used as received. Allsolvents were purified by using standard methods in the art, unlessotherwise indicated.

The reactions set forth below were performed under a positive pressureof nitrogen, argon or with a drying tube, at ambient temperature (unlessotherwise stated), in anhydrous solvents, and the reaction flasks arefitted with rubber septa for the introduction of substrates and reagentsvia syringe. Glassware was oven-dried and/or heat-dried. Analyticalthin-layer chromatography was performed on glass-backed silica gel 60 F254 plates (E Merck (0.25 mm)) and eluted with the appropriate solventratios (v/v). The reactions were assayed by TLC and terminated as judgedby the consumption of starting material.

The TLC plates were visualized by UV absorption or with a p-anisaldehydespray reagent or a phosphomolybdic acid reagent (Aldrich Chemical, 20 wt% in ethanol) which was activated with heat, or by staining in an iodinechamber.

Work-ups were typically done by doubling the reaction volume with thereaction solvent or extraction solvent and then washing with theindicated aqueous solutions using 25% by volume of the extraction volume(unless otherwise indicated). Product solutions were dried overanhydrous sodium sulfate prior to filtration, and evaporation of thesolvents was under reduced pressure on a rotary evaporator and noted assolvents removed in vacuo.

Flash column chromatography [Still et al, J. Org. Chem., 43, 2923(1978)] was conducted using E Merck-grade flash silica gel (47-61 mm)and a silica gel:crude material ratio of about 20:1 to 50:1, unlessotherwise stated. Hydrogenolysis was done at the pressure indicated orat ambient pressure.

¹H NMR spectra were recorded on a Bruker instrument operating at 400MHz, and ¹³C-NMR spectra was recorded operating at 100 MHz. NMR spectrawere obtained as CDCl₃ solutions (reported in ppm), using chloroform asthe reference standard (7.27 ppm and 77.00 ppm) or CD₃OD (3.4 and 4.8ppm and 49.3 ppm), or an internal tetramethylsilane standard (0.00 ppm)when appropriate. Other NMR solvents were used as needed. When peakmultiplicities are reported, the following abbreviations are used:s=singlet, d=doublet, t=triplet, m=multiplet, br=broadened, dd=doubletof doublets, dt=doublet of triplets. Coupling constants, when given, arereported in Hertz. Mass spectra were obtained using LC/MS either in ESIor APCI. All melting points are uncorrected. All final products hadgreater than 90% purity (by HPLC at wavelengths of 220 nm and 254 nm).

The following synthetic examples are intended to illustrate one methodof synthesising the compound of the invention and are not to beconstrued as being limitations thereto.

Example 1 Synthesis of the Compound of the Invention Synthesis of2,6-dichloro-9-isopropyl-9H-purine

2,6-Dichloropurine (2 mmol), isopropanol (8 mmol) and triphenylphosphine(4 mmol) were taken up in 40 ml anhydrous tetrahydrofuran anddiisoproplyazidodicarboxylate (4 mmol) was added drop wise at roomtemperature over a period of 30 min. The reaction mixture was thenstirred at room temperature for a further 24 h. The reaction wasperiodically monitored by TLC or LC-MS. The reaction mixture was pouredin to a beaker containing ice-cold water. Extraction of the aqueouslayer, using 3×100 ml portions of ethyl acetate, afforded the crudeproduct. This was purified by chromatography on a silica gel column(10-80% ethyl acetate in petroleum ether, gradient elution), to give2,6-dichloro-9-isopropyl-9H-purine in a yield of 77%.

Synthesis of 5-(2-chloro-9-isopropyl-9H-purin-6-yl)-pyrimidin-2-ylamine

To a solution of 2,6-dichloro-9-isopropyl-9H-purine (5.21 mmol),5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-ylamine (5.21 mmol) and1,1′-bis(diphenylphosphino) ferrocene palladium (II) chloride complexedwith dichloromethane (0.26 mmol) in peroxide free dioxane (40 ml) wasadded a 2M aqueous solution of sodium carbonate (15.6 mmol). Theresulting mixture was degassed and purged with nitrogen. This reactionmixture was then stirred while being heated on an oil bath maintained at80° C. for 3 h. The reaction was monitored by LC-MS for thedisappearance of the starting purine.

The reaction mixture was cooled to room temperature and the solventsremoved under reduced pressure. The residue was taken up in a mixture ofethyl acetate and water. The organic phase was separated and the aqueouslayer further extracted with 3×100 ml portions of ethyl acetate. Theorganics were dried over sodium sulfate and the solvents removed undervacuum to give5-(2-chloro-9-isopropyl-9H-purin-6-yl)-pyrimidin-2-ylamine in 55% yield.

Synthesis of5-(9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylamine

To a solution of5-(2-chloro-9-isopropyl-9H-purin-6-yl)-pyrimidin-2-ylamine (2.84 mmol)in dimethyl acetamide (18 ml) was added morpholine (2.84 mmol). Thereaction mixture was stirred while being heated on an oil bathmaintained at 94° C. for 12 h. The reaction was monitored for theabsence of starting material by LC-MS. The crude material was directlyloaded onto a preparative HPLC column and purified by chromatography togive 5-(9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylaminein a yield of 58%. ¹H NMR, DMSO-d6: 9.53 (s, 2H), 8.32 (s, 1H), 7.30(bs, 2H), 4.72 (m, 1H), 3.78 (m, 4H), 3.73 (m, 4H), 1.55 (d, 6H). m/z:341.17 [MH]⁺.

Synthesis of5-(8-bromo-9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylamine

To a solution of5-(9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylamine,(1.03 g, 3.03 mmol) in 15 ml of chloroform, was added slowly NBS (594mg, 3.34 mmol) at a temperature of 5° C. The reaction was continued for2 h at this temperature. After simple work-up, the product5-(8-bromo-9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylaminewas purified by flash column (solvent system: 50% ethyl acetate inhexane) to deliver5-(8-bromo-9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylaminein a yield of 52% (660 mg). ¹H NMR, MeOD: 9.67 (s, 2H), 4.90 (m, 1H),3.89 (m, 4H), 3.82 (s, 4H), 1.72 (d, 6H). m/z: 419.31, 421.07 [MH]⁺.

Synthesis of5-(9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylamine

To a solution of5-(8-bromo-9-isopropyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylamine,(30 mg, 0.072 mmol) and Pd(dppf)Cl₂, (3 mg, 5% mmol) in 3 ml ofanhydrous dioxane, was added slowly dimethyl zinc (210 μl, 1.0 M inheptane solution) in a sealed tube. The mixture was heated to about 65°C. MeOH was added drop wise and the solvents removed in vacuo. EtOAc wasadded to the residue and the resulting solution washed with 1 M HCl,water, brine and then dried over Na₂SO₄. The solvent was removed and thecrude mixture was subjected to flash chromatography on silica gel toobtain5-(9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylamine8 mg in a yield of 47%. ¹H NMR, MeOD: 9.40 (s, 2H), 4.81 (m, 1H), 3.89(m, 4H), 3.82 (s, 4H), 3.71 (s, 3H), 1.73 (d, 6H). m/z: 355.16 [MH]⁺.

Example 2 Comparative Biological Testing

The compound of the invention was compared to a number of compoundssynthesised and disclosed in PCT/SG2008/000379 on a number of biologicalparameters.

The parameters tested were:

-   -   Activity in an mTOR assay;    -   Activity in a PI3K assay;    -   Solubility assay    -   Human microsomal stability assay        Details of the methodology of each of these tests are detailed        below:        mTOR Assay

Truncated mTOR kinase and His-tagged 4eBP1 were produced in-house.[γ³³P]-ATP was purchased from Amersham (GE Healthcare). All chemicals,unless otherwise stated, were from Sigma-Aldrich.

Phosphorylation assays were initially performed in a final volume of 20μL in 384-well polypropylene plate (Greiner). Compounds were typicallytested over the range from 100 μM to 0.006 μM, in 8 step dilutions, induplicate. 10 μL/well of 2× Enzyme-Substrate solution (1.5 μg/mL mTOR,40 μg/mL 4eBP1 in 1× assay buffer: 10 mM Hepes pH 7.5, 50 mM NaCl and 10mM MnCl₂) were first added to the sample plate containing 1 μL/well oftest compound in neat DMSO. The reaction was initiated by adding 10μL/well of 20 μM ATP solution (final assay concentration 10 μM ATP and0.4 μCi/well of [γ³³P]-ATP). After 1 hour incubation at roomtemperature, the reaction was terminated with 40 μL/well of 20 mM EDTA/1mM ATP solution.

50 μL/well of the stopped reaction mix was then transferred to a384-well MultiScreenHTS-PH filter plate (Millipore) pre-added with 50μL/well of 1% phosphoric acid. The plate was washed 4 times with 120μL/well of 0.5% phosphoric acid via vacuum filtration. Finally, 10μL/well of Optiphase™ SuperMix liquid scintillation cocktail (PerkinElmer) was added. After minimum 1 hour of incubation, counting wasperformed in a Wallac MicroBeta TriLux scintillation counter usingcoincidence counting mode with crosstalk correction. IC₅₀ is defined asthe concentration of compound required for 50% of maximum possibleinhibition of kinase enzyme activity.

PI3K Assay

Recombinant PI3K p110α/p85 was prepared in-house. Phosphatidylinositol(PtdIns), phosphotidylserine (PtdSer) and all other unspecifiedchemicals were purchased from Sigma-Aldrich. [γ³³P]ATP and Optiphasescintillant were obtained from Perkin Elmer.

Assays were performed in a final assay volume of 25 μL in 384-wellMaxisorp plates (Nunc). Compounds were tested at 8 concentrations in3-fold serial dilution, generally starting from 10 μM. Maxisorp plateswere coated with 20 μL/well of a 1:1 mixture of PtdIns and PtdSer [0.1mg/mL each dissolved in chloroform:ethanol (3:7)] and left overnight ina fume hood at room temperature (RT) to dry.

The enzyme reaction was created by pipetting 5 μL/well of compound (in2.5% DMSO), 10 μL/well of enzyme (0.5 μg/mL p110α+1 μg/mL p85), and 10μL/well of 5 μM ATP with 5 μCi/mL [γ³³P]ATP in assay buffer (finalconcentrations: 0.2 μg/mL p110α, 2 μM ATP, 0.05 μCi/well [γ³³P]ATP in 1×assay buffer: 100 mM Tris-HCl pH 7.0, 200 mM NaCl, 8 mM MgCl₂). Thereaction was incubated for 1 hour at RT and terminated with 30 μL/wellof 50 mM EDTA solution. The plate was then washed twice with TBS, dried,and added with 30 μL/well of scintillant before it was counted in aMicroBeta Trilux. IC₅₀ is defined as the concentration of compoundrequired for 50% of maximum possible inhibition of kinase enzymeactivity.

Microsomal Stability Assay

Compound stability is initially assessed in vitro using a highthroughput format in 96 well plates (Whatman) involving incubation withhuman liver microsomes (HLM). Verapamil, purchased from Sigma-Aldrich,is used as a reference standard in the assay. HLM are purchased fromXeno Tech (20 mg/mL in 250 mM sucrose solution). A 100 mM stock solutionof potassium phosphate buffer is pre-prepared by combining 80 mL of 1MK₂HPO₄ and 20 mL of 1 M KH₂PO₄ in 900 mL of water (pH adjusted to 7.4using diluted HCl) and stored at room temperature. K₂HPO₄.3H₂0 andKH₂PO₄ are obtained from Sigma Aldrich and the NADPH Regeneration SystemSolutions A and B from Gentest. The stop solution used to quench thereaction is a pre-prepared mixture of acetonitrile and DMSO (80:20) andis stored at 4° C. All solvents used are HPLC grade and the water usedduring the stock solution preparation and during LC-MS analysis isdeionised using a Milli-Q system.

2.5 μL of a 10 mM stock solution of the test compound in DMSO is diluted200 times by mixing with 500 μL of a 50 mM potassium phosphate buffer(pH 7.4, prepared by dilution of the 100 mM stock buffer solution withwater) to give 500 μL of a 50 μM solution. 8 μL of the compound mix isthen added to 72 μL of a pre-prepared incubation mix made up of water(2250 μL), 100 mM potassium phosphate buffer (2900 μL), NADPHRegeneration System Solution B (58 μL), NADPH Regeneration SystemSolution A (290 μL) and HLM (250 μL). The resulting reaction mixture(final compound concentration of 5 μM) is then incubated at 37° C. in aB. Braun Certomat H incubator after which a 50 μL aliquot is dispensedto a well on a separate plate well containing 100 μL of Stop solution.After centrifugation at 4° C. for 15 min at 2000 rpm a 100 μL sample ofthe resulting supernatant is transferred to an LC-MS plate for analysis.Each test compound is sampled multiple times and incubated for a seriesof time points (5, 15, 30, 45 and 60 min). Remaining compoundconcentration is determined by LC-MS (ABI Qtrap 3200) and comparison toa reference solution of known concentration. Stability is then expressedas a half-life in minutes (t½).

High Throughput Solubility Assay

Compound solubility is determined in a high-throughput kineticsolubility profiling method using a 96-well format. Compound solubilityis assessed using a UV/Visible Microplate Spectrophotometer (MolecularDevices SpectraMax Plus384). Vorinostat (SAHA) and Nicardipine,purchased from Sigma, are used as reference standards.

Compounds in DMSO are diluted with phosphate buffer (Sigma) at a finalconcentration of 250 μM (5 μL of a 10 mM stock solution in 195 μLphosphate buffer pH 7) and thoroughly mixed. The mixture is then shakenat 600 rpm for 1.5 h and allowed to stand at room temperature for 2 h.The plate is then centrifuged at 1500 g for 15 min. The resultingsupernatant (80 μL) is transferred to the UV-analysis plate and dilutedwith DMSO (20 μL). The samples are quantified using calibration stocksof the respective compound made in phosphate buffer/DMSO (80:20).

The compounds tested were as follows:

The results of the biological testing are summarised in table 1.

TABLE 1 Cmpd of Cmpd A Cmpd B Cmpd C Cmpd D Cmpd E the invention mTOR 21nM 16 nM 27 nM 40 nM 122 nM 36 nM PI3K 43 nM 17 nM 16 nM 15 nM 18 nM 11nM Solubility 44 μM 18 μM >250 μM >250 μM 35 μM 178 μM T½ HLM >60min >60 min 36 min 26 min >60 min >60 min

As can be seen whilst all compounds had some degree of activity thecompound of the invention had activity against both the enzymes ofinterest comparable to the activity of all the comparator compoundsexcept compound E which had significantly lower activity against mTOR.As such all the compounds were potential drug candidates althoughcompound E was somewhat less active against mTOR.

Not all the compounds tested displayed acceptable solubilitycharacteristics, however. For example the low solubility results forcompounds A, B and E meant that these compounds would not make good drugcandidates. Their low solubilities make them difficult to formulateeffectively in physiologically acceptable carriers hence reducing thelikelihood that they would have good oral pharmacokinetics in humans. Incontrast the solubility of compounds C, D and the compound of theinvention were acceptable for drug candidate compounds.

In relation to in vitro metabolic stability, however, the results weresignificantly different. In these tests compounds A, B, E and thecompound of the invention had acceptable stability in human livermicrosome studies. This suggests that if these compounds could besuccessfully administered then they would be sufficiently stable toachieve the desired physiological effect in the patient. Moreover of thecompounds which exhibit both good mTOR/PI3K inhibitory activity and goodsolubility (C,D and the compound of the invention), the only one withacceptable metabolic stability is the compound of the invention.

All the compounds A-E and the compound of the invention show goodactivity in these assays but the compound of the invention is one of thestrongest due to its combination of potent target inhibition activity,good aqueous solubility and good metabolic stability.

In summary the biological results achieved for the compounds tested inthe biological studies conducted above suggest that notwithstanding theapparently close similarity of the compound of the invention with anumber of the comparator test compounds the compound of the invention isthe only one that demonstrates the required combination of activity,sufficient aqueous solubility and metabolic stability to suggest thatthe compound has application as a drug moiety. These studies thereforedemonstrate the superiority of this compound as a drug candidate.

Example 3 Cell-Based Efficacy Biomarker Assays (pp70-S6KT389, pAktS473)

In order to further demonstrate the efficacy of the compound of theinvention two cell based biomarker assays were conducted on the compoundof the invention. The methodology was as follows:

AlphaScreen® SureFire p-Akt (Ser 473) 384 Kit (TGR, Cat. No.: TGRAS500),

AlphaScreen® SureFire phospho-p70 S6 kinase (Thr 389) 384 kit (TGR, Cat.No.:TGR70S500) and Proxiplate-384 Plus (Perkin Elmer, Cat. No.: 6008280)were purchased from Perkin Elmer. The human prostatic carcinoma cellline (PC-3) was purchased from ATCC. All chemicals, unless otherwisestated, were from Sigma-Aldrich.

On Day 1, 200 μl of a 2×105 cells/ml cell solution of PC3 cells wereseeded into each well of a 96-well plate. Compounds were added 24 hafter seeding and were typically tested over a range from 10 μM to 4.6nM, in 8 step dilutions, in triplicate. The final concentration of DMSOduring the 4 h incubation step at 37° C. was 0.1%. After the incubationstep, the supernatant was removed, the cells were lysed with 1× Lysisbuffer (provided by the AlphaScreen kit) and gently shaken for 10 min. 4μl of the lysates and 5 μl of the Reaction Buffer plus Activation buffermix containing AlphaScreen Acceptor beads were added into each 384-well(Ratio of Reaction Buffer:Activation Buffer:Acceptor Beads is 40:10:1)and gently shaken for 2 h (room temperature, dark). 2 μl of Dilutionbuffer containing AlphaScreen® Donor beads (Ratio of DilutionBuffer:Donor Beads is 20:1) were added into each well on a 384-plate,placed on a plate shaker for 1-2 mins and incubated at room temperatureovernight.

A BMG Pherostar plate reader was used to read the 384-well plate usingstandard AlphaScreen settings (Measurement mode: Alphascreen; readingmode: Endpoint; optic mode: AlphaScreen 680 570; Position delay: 0.10 s;Excitation time: 0.30 s; Integration start: 0.34 s; integration time:0.30 s; Gain: 3000).

IC₅₀ is defined as the molar concentration of a compound, which produces50% of the maximum possible inhibition of kinase enzyme activity by thiscompound. The IC₅₀ of5-(9-isopropyl-8-methyl-2-morpholin-4-yl-9H-purin-6-yl)-pyrimidin-2-ylaminefor inhibition of of phosphorylation of p70-S6KT389 and pAktS473 wasfound to be 24 nM and 9 nM respectively.

The biomarker results demonstrate the efficacy of the compound of theinvention in the inhibition of kinase enzyme activity.

What is claimed is:
 1. A pharmaceutical composition comprising acompound of formula (I):

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable diluent, excipient or carrier.
 2. A method of inhibiting aprotein kinase selected from the group consisting of a serine/threonineprotein kinase or a fragment or a complex thereof or a functionalequivalent thereof and a PI3 kinase or a fragment or a complex thereofor a functional equivalent thereof, the method including exposing theprotein kinase or a fragment or complex thereof or a functionalequivalent thereof and/or co-factor(s) thereof to an effective amount ofa compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein theserine/threonine protein kinase or fragment or complex thereof is anmTOR protein kinase, or a fragment thereof, or a complex thereof or afunctional equivalent thereof.
 3. The method according to claim 2,wherein the protein kinase is an mTOR protein kinase, or a fragmentthereof, or a complex thereof or a functional equivalent thereof.
 4. Themethod according to claim 3, wherein the mTOR protein kinase or fragmentor complex or functional equivalent thereof is selected from the groupconsisting of mTORC1 or a fragment or complex thereof or a functionalequivalent thereof and mTORC2 or a fragment or complex thereof or afunctional equivalent thereof.
 5. The method according to claim 2wherein the protein kinase is a PI3K kinase or a fragment thereof or acomplex thereof or a functional equivalent thereof.
 6. The methodaccording to claim 5 wherein the PI3 kinase or a fragment thereof or acomplex thereof or a functional equivalent thereof, is a class I PI3K ora fragment thereof or a complex thereof or a functional equivalentthereof.